Diagnostic preparation for detection of pseudomonas



United States Patent 3,122,489 DEAGNGSTHI PREPARATEON FOR DETECTEON 0F PSEUDUMONAS Frank J. Turner, Succasunna, and Glenvilie R. Watson,

East Grange, NIL, assignors to Warner-=Lambert Pharmaceutical tCompany, Morris Plains, NJL, a corporation of Delaware No Drawing. Filed May 25, 1961, Ser. No. 112,536 3 Claims. (Cl. 167-84.5)

The present invention relates to a new and novel diagnostic preparation adapted to the rapid and positive identification of the genus Pseudomonas in biological sources.

Various species of the genus Pseudomonas, for example Pseudomonas aeruginosa, are known to be responsible for many types of infections in man, for example infections of the urinary tract, ear and skin. Heretofore, positive identification of the organism causing such infections has proved difficult, due to the fact that the Pseudomonas genus is not readily distinguishable. The test procedures heretofore available to detect the presence of Pseudomonas involve the use of unstable liquid reagent systems which must be prepared immediately prior to each determination. There has long been a need for a stable diagnostic preparation which will permit a rapid and positive identification of Pseudomonas without the necessity of formulating a fresh reagent system immediately prior to the test.

it is, therefore, an important object of this invention to provide a diagnostic preparation which permits the rapid and positive identification of Pseudomonas.

Another object of this invention is to provide a diagnostic preparation for the detection of Pseudomonas which is stable for long periods of time.

Other objects and the advantages of this invention will become apparent from the following detailed description.

V/e have now found that the aforementioned objects are fulfilled by providing a strip of a bibulous material which is impregnated over at least a portion of its area with a dried reagent system comprising an N-lower alkyl derivative of p-phenylenediamine or an acid addition salt thereof and a hydroxy substituted naphthalene derivative.

As a carrier for the reagent system, a bibulous material is employed, that is, any porous, absorbent sheet material such as paper. The bibulous material is normally cut into narrow strips to facilitate packaging in small bottles. The reagent system is applied either to the entire strip or a portion thereof and is preferably applied to several spaced portions of the strip so that each strip may be used for several tests.

The reagent system applied to the bibulous material comprises a hydroxy substituted naphthalene derivative and an N-lower alkyl derivative of p-phenylenediamine,

having the formula:

wherein R is lower alkyl and R R and R are hydrogen or lower alkyl, the term lower alkyl referring to straight or branched chain aliphatic groups containing 1 to 6 carbon atoms, or an acid addition salt of said N- lower alkyl derivative of p-phenylenediamine.

The hydroxy substituted naphthalene derivatives which are useful in the preparation of our diagnostic preparation are monoor di-hydroxy naphthalenes which may in addition contain up to 3 sulfonic acid groups in the molecule, for example oc-IIQPhthOl, fi-naphthol, 1,3-dihydroxynaphthalene, 1,S-dihydroxynaphthalene, 4,5-dihy droxynaphthalene, 2 hydroxynaphthalene-3,6-disulfonic acid, 1-hydroxynaphthalene-Z-sulfonic acid, 1,8-dihy- 3,122,48fi Patented Feb. 25, 1964 "ice I 2 doxynaphthalene-3,6-disulfonic acid, 4,5-dihydroxynaphthalene-2,7-disulfonic acid and the like.

Useful p-phenylenediamine derivatives include N-methyl p-phenylenediamine, N,N-dimethyl-p-phenylenediamine, tetramethyl-p-phenylenediamine, N-ethyl-p-phenylenediamine and the like. It is preferred that the p-phenylenediamine derivative be present in our composition in the form of an acid addition salt such as the hydrochloride, maleate or oxalate. We have found that a reagent system comprising u-naphthol and N,N-dimethyl-p-phenylenediamine oxalate is particularly useful in the formation of our diagnostic preparation.

The reagent system is applied to the bibulous material in the form of solutions of each ingredient in a volatile solvent and the impregnated material is then dried to evaporate the solvents. Normally, solutions containing about 0.5 to about 2.5 percent by weight of each ingredient are used. It has been found that the reagent system should be applied in the relative proportions of about 0.5 to about 1 part by weight of said hydroxy substituted naphthalene derivative for each part by weight of said pphenylenediamine derivative employed.

The diagnostic preparation of our invention consists of the bibulous material with the dried reagent system applied thereto. While this preparation is stable and represents a marked improvement over the unstable systems previously used, we have found that greatly enhanced stability is obtained by the incorporation into the reagent system of about 0.2 to about 1 part by weight of cysteine per part by weight of said p-phenylenediamine derivative. The presence of cysteine imparts an unusual resistance to decomposition of the ingredients of the reagent and insures a diagnostic preparation which is effective for many months after its manufacture.

in use, a loopful of an agar culture or a centrifuged liquid culture of the unknown organism, for example, a culture obtained from a urine specimen or the discharge from a suspected lesion, is placed upon an impregnated area of the bibulous material. If Pseudomonas are present in the culture, a color develops in the bibulous material within less than a minute, thus affording a simple means of detecting the presence of Pseudomonas in the unknown culture. I

The following examples are included in order further to illustrate the present invention:

Example I Two parts of a 1 percent by weight solution of a-naphthol in percent ethanol are added to three parts of a 1 percent by weight aqueous solution of N,N-dimethyl-pphenylenediamine oxalate and drops of the resulting mixture are placed at /2 inch intervals along the length of a filter paper strip which is 6 inches long by inch wide. The strip is permitted to dry at room temperature and is stored over silica gel.

The application of a loopful of an agar culture or centrifuged liquid culture of the suspect organism to one of the dried spots on the filter paper strip results in the development of a bright permanent blue color if Pseudomonas are present.

Example II Strips are prepared as described in Example I except that the aqueous solution of N,N-dimethyl-p-phenylenediamine oxalate employed contains 0.56 percent by weight of cysteine.

The resulting impregnated and dried strips are employed in the manner described but are found to have an enhanced stability compared to the strips of Example I.

It is understood that the foregoing detailed description is given merely by way of illustration and that many vara iations may be made therein without departing from the spirit of our invention.

Having described our invention, What we desire to secure by Letters Patent is:

1. A diagnostic preparation for the rapid and positive detection of Pseudornonas in an unknown culture which comprises a strip of a bibulous material impregnated with a dry reagent system comprising about 0.5 to about 1 part by weight of a-naphthol and about 1 part by weight of an acid addition salt of N,N-dimethyl-p-phenylenediamine.

2. A diagnostic preparation for the rapid and positive detection of Pseudomonas in an unknown culture which comprises a strip of a bibulous material impregnated with a dry reagent system comprising about (2.5 to about 1 part by weight of a-naphthol and about 1 part by Weight of N,N-dirnethyl-p-phenylenediamine oxalate.

3. A diagnostic preparation according to claim 2 wherein said reagent system includes about 0.2 to about 1 part by weight of cysteine.

References Cited in the fiie of this patent Physician Desk Reference, 1960, pp. 616-617. Physician Desk Reference, 1961, p. 517.

Kurachi: Chem. A bst., vol. 54, 1960, pp. 7824-7825. Rogoff: Chem. Abst, vol. 53, 1959, p. 18168(a). Burton: Chem. Abst., vol. 42, 1948, p. 945(a). 

1. A DIAGNOSTIC PREPARATION FOR THE RAPID AND POSITIVE DETECTION OF PSEUDOMONAS IN AN UNKNOWN CULTURE WHICH COMPRISES A STRIP OF A BIBNULOUS MATERIAL IMPREGNATED WITH A DRY REAGENT SYSTEM COMPRISING ABOUT 0.5 TO ABOUT 1 PART OF WEIGHT OF A-NAPHTHOL AND ABOUT 1 PART BY WEIGHT OF AN ACID ADDITION SALT OF N,N-DIMETHYL-P-PHENYLENEDIAMINE. 